Computational protocol: Transcriptome-wide selection of a reliable set of reference genes for gene expression studies in potato cyst nematodes (Globodera spp.)

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Protocol publication

[…] This study took advantage of a recently published transcriptome dataset from a G. rostochiensis hatching experiment conducted by our team []. These data (NCBI bioproject accession number PRJNA274143) include sampling at different life stages, including dry cyst, hydrated cyst, hydrated cyst soaked in PRD for 1 h, 8 h, 24 h, 48 h, and 7 d, and fully hatched infective larvae (J2). Library preparation and sequencing were performed at McGill University and Génome Québec Innovation Centre (Montreal, Quebec, Canada) using the TruSeq RNA sample prep kit v2 (Illumina) and a HiSeq 2000 sequencer (Illumina, San Diego, California, United States). All eight samples were multiplexed and sequenced in one lane for 100 bp paired-end reads. Two replicates were processed and assembled into a de novo transcriptome using the Trinity assembler (for details, see Duceppe et al. []). [...] Primers were designed using PrimerQuest tool (Integrated DNA Technologies, Inc., Coralville, Iowa, United States) based on the sequences retrieved from the G. rostochiensis RNA-Seq dataset. Target fragments lengths were designed between 84 and 130 bp. Primer information for the candidate reference genes is listed in . Reactions were prepared using QuantiTect SYBR Green PCR kit (Qiagen) and amplified on a Mx3000P qPCR System (Agilent Technologies) in a final volume of 20 μL according to the manufacturer’s instructions. Melting curve analyses were done following the amplification cycles in order to examine the specificity of the reactions. Amplification efficiencies were calculated with dry cysts using the Real-time PCR Miner algorithm (ver. 4.0) []. [...] RefFinder [], a wrapper tool that integrates the statistical algorithm of BestKeeper [], NormFinder [], and geNorm [], and the ΔCt method [], was used to compare and rank the tested candidate reference genes. Based on the rankings from each program, it assigns an appropriate weight to an individual gene and calculates the geometric mean of their weights for the overall comprehensive ranking. Gene expressions from both the RNA-Seq database (read numbers) and qRT-PCR data (Cq values) across all treatments were compared. The variability of each gene across all treatments and replicates was also directly observed by plotting the distribution of the raw Cq values from the qRT-PCR experiment. […]

Pipeline specifications

Software tools Trinity, PrimerQuest, BestKeeper, NormFinder
Applications RNA-seq analysis, qPCR
Organisms Solanum tuberosum, Caenorhabditis elegans
Diseases Nematode Infections